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Melanoma is a cancer of melanocytes, with multiple subtypes based on body site location. Cutaneous melanoma is associated with skin exposed to ultraviolet radiation; uveal melanoma occurs in the eyes; mucosal melanoma occurs in internal mucous membranes; and acral melanoma occurs on the palms, soles, and nail beds. Here, we present the largest whole-genome sequencing study of melanoma to date, with 570 tumors profiled, as well as methylation and RNA sequencing for subsets of tumors. Uveal melanoma is genomically distinct from other melanoma subtypes, harboring the lowest tumor mutation burden and with significantly mutated genes in the G-protein signaling pathway. Most cutaneous, acral, and mucosal melanomas share alterations in components of the MAPK, PI3K, p53, p16, and telomere pathways. However, the mechanism by which these pathways are activated or inactivated varies between melanoma subtypes. Additionally, we identify potential novel germline predisposition genes for some of the less common melanoma subtypes. Significance: This is the largest whole-genome analysis of melanoma to date, comprehensively comparing the genomics of the four major melanoma subtypes. This study highlights both similarities and differences between the subtypes, providing insights into the etiology and biology of melanoma. This article is highlighted in the In This Issue feature, p. 2711

Supplementary Data from Comparative Genomics Provides Etiologic and Biological Insight into Melanoma Subtypes

Melanoma is a cancer of melanocytes, with multiple subtypes based on body site location. Cutaneous melanoma is associated with skin exposed to ultraviolet radiation; uveal melanoma occurs in the eyes; mucosal melanoma occurs in internal mucous membranes; and acral melanoma occurs on the palms, soles, and nail beds. Here, we present the largest whole-genome sequencing study of melanoma to date, with 570 tumors profiled, as well as methylation and RNA sequencing for subsets of tumors. Uveal melanoma is genomically distinct from other melanoma subtypes, harboring the lowest tumor mutation burden and with significantly mutated genes in the G-protein signaling pathway. Most cutaneous, acral, and mucosal melanomas share alterations in components of the MAPK, PI3K, p53, p16, and telomere pathways. However, the mechanism by which these pathways are activated or inactivated varies between melanoma subtypes. Additionally, we identify potential novel germline predisposition genes for some of the less common melanoma subtypes.Significance:This is the largest whole-genome analysis of melanoma to date, comprehensively comparing the genomics of the four major melanoma subtypes. This study highlights both similarities and differences between the subtypes, providing insights into the etiology and biology of melanoma.This article is highlighted in the In This Issue feature, p. 2711

The incidence of oesophageal adenocarcinoma (OAC) is rising in Western countries, with a 5-year overall survival (OS) rate of 14%. Curative treatment based on oesophagectomy is only suitable for ~50% of patients due to late-stage diagnosis. While the addition of preoperative chemotherapy or chemoradiotherapy has improved OS in OAC patients, little is known about the molecular basis of treatment response and patient outcomes. We investigated multi-omics data including whole-genome sequencing, RNA sequencing, methylation profiles and immunohistochemistry from 115 OAC patients mostly from DOCTOR phase II clinical trial that utilized neoadjuvant therapy (ANZCTR-ACTRN12609000665235). We identified genomic features associated with poor OS, such as the APOBEC mutational signature. We also showed that positron emission tomography non-responders have more sub-clonal genomic copy number alterations. RNA sequencing and methylation profiles suggested four immune clusters associated with OS and progression-free survival. The immune-suppressed cluster was associated with worse survival and epithelial-mesenchymal transition signature and enriched with myeloid-derived cells. The immune hot cluster was associated with better survival and enriched with T-cells, myeloid-derived cells, interferon-gamma and alpha responses, and immune markers such as CCL5, CD8A, and NKG7. We investigated multi-omic features of OAC tumours from patients who were part of phase II clinical trial. We discovered prognostic features such as mutational signatures and complex genomic events. We identified distinct immune clusters associated with patient outcomes and the potential of immunotherapy for select clusters. We characterized molecular features of OAC patients which has the potential to predict responses to neoadjuvant therapy and develop better-selected therapy, monotherapy, or combination therapy in the future.

Approximately 10-15% of breast cancers are associated with a strong family history of disease. Pathogenic variants in BRCA1, BRCA2 or other moderate to highly penetrant susceptibility genes (e.g. TP53, ATM, CHEK2, PALB2 and PTEN) account for a number of breast cancer families. However, for over 50% of families the underlying genetic contribution to their risk remains unknown (termed here as non-BRCA1/2). This has a profound impact for how individuals and their families are managed in the clinic. We applied whole genome sequencing (WGS) to determine whether somatic mutation analysis can reveal insight into the aetiology of familial breast cancer. The full repertoire of somatic mutations was evaluated in 26 BRCA1, 22 BRCA2 and 32 non-BRCA1/2 tumours; including SNPs, indels, copy number changes and structural rearrangements, and mutational signatures. Genomes were also analysed using the HRD Index and HRDetect, as predictors of homologous recombination deficiency. BRCA1, BRCA2 and non-BRCA1/2 tumours exhibited a different burden of mutations, a different spectrum of mutational signatures and different telomere length. Based on collective patterns of mutation signatures, tumours were classified as 'BRCA1-like', 'BRCA2-like' or 'non-BRCA1/2-like' with a 15% rate of tumour re-classification from their original clinical BRCA status. The results demonstrate the power of WGS to differentiate between BRCA1 and BRCA2 driven tumours; in the identification of double-pathogenic germline mutation carriers based on the resulting somatic mutation signature; and in the interpretation of BRCA unclassified variants. WGS of tumour genomes reveals fascinating insights into tumour aetiology and could compliment current genetic testing of breast cancer families. Citation Format: Simpson P, Nones K, Johnson J, Newell F, Patch A-M, Thorne H, Kazakoff S, De Luca X, Parsons M, Ferguson K, Reid L, McCart Reed A, Srihari S, Lakis V, Davidson A, Mukhopadhyay P, Holmes O, Xu Q, Wood S, Leonard C, Beasley J, Degasperi A, Nik-Zainal S, Ragan M, Spurdle A, Khanna KK, Lakhani S, Pearson J, Chenevix-Trench G, Waddell N. Using whole genome sequencing and somatic mutation signatures to unravel insight into familial breast cancer aetiology [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr P5-10-01.

BackgroundEndometrial cancer (EC) is a major gynecological cancer with increasing incidence. It comprised of four molecular subtypes with differing etiology, prognoses, and response to chemotherapy. In the future, clinical trials testing new single agents or combination therapies will be targeted to the molecular subtype most likely to respond. Pre-clinical models that faithfully represent the molecular subtypes of EC are urgently needed, we sought to develop and characterize a panel of novel EC patient-derived xenograft (PDX) models.MethodsHere, we report whole exome or whole genome sequencing of 11 PDX models and the matched primary tumor. Analysis of multiple PDX lineages and passages was performed to study tumor heterogeneity across lineages and/or passages. Based on recent reports of frequent defects in the homologous recombination (HR) pathway in EC, we assessed mutational signatures and HR deficiency scores and correlated these with in vivo responses to the PARP inhibitor (PARPi) talazoparib in six PDXs representing the different molecular subtypes of EC.ResultsPDX models were successfully generated from all four molecular subtypes of EC and uterine carcinosarcomas, and they recapitulated morphology and the molecular landscape of primary tumors without major genomic drift. We also observed a wide range of inter-tumor and intra-tumor heterogeneity, well captured by different PDX lineages, which could lead to different treatment responses. An in vivo response to talazoparib was detected in two p53mut models consistent with stable disease, however both lacked the HR deficiency genomic signature.ConclusionsEC PDX models represent the four molecular subtypes of disease and can capture intra-tumoral heterogeneity of the original primary tumor. PDXs of the p53mut molecular subtype showed sensitivity to PARPi, however, deeper and more durable responses will likely require combination of PARPi with other agents.

Here we report the DNA methylation profile of 84 sporadic pancreatic neuroendocrine tumors (PanNETs) with associated clinical and genomic information. We identified three subgroups of PanNETs, termed T1, T2 and T3, with distinct patterns of methylation. The T1 subgroup was enriched for functional tumors and ATRX, DAXX and MEN1 wild-type genotypes. The T2 subgroup contained tumors with mutations in ATRX, DAXX and MEN1 and recurrent patterns of chromosomal losses in half of the genome with no association between regions with recurrent loss and methylation levels. T2 tumors were larger and had lower methylation in the MGMT gene body, which showed positive correlation with gene expression. The T3 subgroup harboured mutations in MEN1 with recurrent loss of chromosome 11, was enriched for grade G1 tumors and showed histological parameters associated with better prognosis. Our results suggest a role for methylation in both driving tumorigenesis and potentially stratifying prognosis in PanNETs., Lakis et al. report novel findings related to the epigenetic landscape of pancreatic neuroendocrine cancer. This relatively large cohort provides functional insights into the epigenetic wiring of pancreatic neuroendocrine tumor sub-types with the potential ability to stratify tumours of different prognosis.

Background Endometrial cancer (EC) is a major gynecological cancer with increasing incidence. It comprises four molecular subtypes with differing etiology, prognoses, and responses to chemotherapy. In the future, clinical trials testing new single agents or combination therapies will be targeted to the molecular subtype most likely to respond. As pre-clinical models that faithfully represent the molecular subtypes of EC are urgently needed, we sought to develop and characterize a panel of novel EC patient-derived xenograft (PDX) models. Methods Here, we report whole exome or whole genome sequencing of 11 PDX models and their matched primary tumor. Analysis of multiple PDX lineages and passages was performed to study tumor heterogeneity across lineages and/or passages. Based on recent reports of frequent defects in the homologous recombination (HR) pathway in EC, we assessed mutational signatures and HR deficiency scores and correlated these with in vivo responses to the PARP inhibitor (PARPi) talazoparib in six PDXs representing the copy number high/p53-mutant and mismatch-repair deficient molecular subtypes of EC. Results PDX models were successfully generated from grade 2/3 tumors, including three uterine carcinosarcomas. The models showed similar histomorphology to the primary tumors and represented all four molecular subtypes of EC, including five mismatch-repair deficient models. The different PDX lineages showed a wide range of inter-tumor and intra-tumor heterogeneity. However, for most PDX models, one arm recapitulated the molecular landscape of the primary tumor without major genomic drift. An in vivo response to talazoparib was detected in four copy number high models. Two models (carcinosarcomas) showed a response consistent with stable disease and two models (one copy number high serous EC and another carcinosarcoma) showed significant tumor growth inhibition, albeit one consistent with progressive disease; however, all lacked the HR deficiency genomic signature. Conclusions EC PDX models represent the four molecular subtypes of disease and can capture intra-tumor heterogeneity of the original primary tumor. PDXs of the copy number high molecular subtype showed sensitivity to PARPi; however, deeper and more durable responses will likely require combination of PARPi with other agents.

To increase understanding of the genomic landscape of acral melanoma, a rare form of melanoma occurring on palms, soles or nail beds, whole genome sequencing of 87 tumors with matching transcriptome sequencing for 63 tumors was performed. Here we report that mutational signature analysis reveals a subset of tumors, mostly subungual, with an ultraviolet radiation signature. Significantly mutated genes are BRAF, NRAS, NF1, NOTCH2, PTEN and TYRP1. Mutations and amplification of KIT are also common. Structural rearrangement and copy number signatures show that whole genome duplication, aneuploidy and complex rearrangements are common. Complex rearrangements occur recurrently and are associated with amplification of TERT, CDK4, MDM2, CCND1, PAK1 and GAB2, indicating potential therapeutic options., Acral melanoma occurs on the soles of the feet, palms of the hands and in nail beds. Here, the authors reports the genomic landscape of 87 acral melanomas and find that some tumors harbor a UV signature and that the tumors are diverse at the levels of mutational signatures, structural aberrations and copy number signatures.

We concurrently examine the whole genome, transcriptome, methylome, and immune cell infiltrates in baseline tumors from 77 patients with advanced cutaneous melanoma treated with anti-PD-1 with or without anti-CTLA-4. We show that high tumor mutation burden (TMB), neoantigen load, expression of IFNγ-related genes, programmed death ligand expression, low PSMB8 methylation (therefore high expression), and T cells in the tumor microenvironment are associated with response to immunotherapy. No specific mutation correlates with therapy response. A multivariable model combining the TMB and IFNγ-related gene expression robustly predicts response (89% sensitivity, 53% specificity, area under the curve [AUC], 0.84); tumors with high TMB and a high IFNγ signature show the best response to immunotherapy. This model validates in an independent cohort (80% sensitivity, 59% specificity, AUC, 0.79). Except for a JAK3 loss-of-function mutation, for patients who did not respond as predicted there is no obvious biological mechanism that clearly explained their outlier status, consistent with intratumor and intertumor heterogeneity in response to immunotherapy.

Background Whole-genome sequencing (WGS) is a powerful method for revealing the diversity and complexity of the somatic mutation burden of tumours. Here, we investigated the utility of tumour and matched germline WGS for understanding aetiology and treatment opportunities for high-risk individuals with familial breast cancer. Patients and methods We carried out WGS on 78 paired germline and tumour DNA samples from individuals carrying pathogenic variants in BRCA1 (n = 26) or BRCA2 (n = 22) or from non-carriers (non-BRCA1/2; n = 30). Results Matched germline/tumour WGS and somatic mutational signature analysis revealed patients with unreported, dual pathogenic germline variants in cancer risk genes (BRCA1/BRCA2; BRCA1/MUTYH). The strategy identified that 100% of tumours from BRCA1 carriers and 91% of tumours from BRCA2 carriers exhibited biallelic inactivation of the respective gene, together with somatic mutational signatures suggestive of a functional deficiency in homologous recombination. A set of non-BRCA1/2 tumours also had somatic signatures indicative of BRCA-deficiency, including tumours with BRCA1 promoter methylation, and tumours from carriers of a PALB2 pathogenic germline variant and a BRCA2 variant of uncertain significance. A subset of 13 non-BRCA1/2 tumours from early onset cases were BRCA-proficient, yet displayed complex clustered structural rearrangements associated with the amplification of oncogenes and pathogenic germline variants in TP53, ATM and CHEK2. Conclusions Our study highlights the role that WGS of matched germline/tumour DNA and the somatic mutational signatures can play in the discovery of pathogenic germline variants and for providing supporting evidence for variant pathogenicity. WGS-derived signatures were more robust than germline status and other genomic predictors of homologous recombination deficiency, thus impacting the selection of platinum-based or PARP inhibitor therapy. In this first examination of non-BRCA1/2 tumours by WGS, we illustrate the considerable heterogeneity of these tumour genomes and highlight that complex genomic rearrangements may drive tumourigenesis in a subset of cases.

Endometrial cancer is a major gynaecological cancer with a high incidence resulting in over 500 Australian women dying every year. Good pre-clinical models are urgently needed to study the biology of this disease, and to develop and test novel treatment strategies. Most current pre-clinical research is conducted in cell lines that often do not resemble the disease on the molecular level and do not recapitulate tumour heterogeneity, which results in untranslatable research findings. Patient-derived xenograft (PDX) models overcome the issues of representing tumour molecular landscape and heterogeneity, making them a translatable pre-clinical cancer model. Here, we generated and genomically characterised 11 PDX models by performing whole genome or whole exome sequencing of primary patient tumours and matched PDX samples. We performed a detailed genomic characterisation of these models to determine their suitability as pre-clinical models, study tumour heterogeneity and identify biomarkers of response and resistance. PDX models were successfully generated from all four molecular subtypes of EC and uterine carcinosarcomas, and they recapitulated morphology and the molecular landscape of primary tumors without major genomic drift. We also observed a wide range of inter-tumor and intra-tumor heterogeneity, well captured by different PDX lineages, which could lead to different treatment responses. An in vivo response to talazoparib was detected in two p53mut models consistent with stable disease, however both lacked the HR deficiency genomic signature. EC PDX models represent the four molecular subtypes of disease and can capture intra-tumoral heterogeneity of the original primary tumor. PDXs of the p53mut molecular subtype showed sensitivity to PARPi, however, deeper and more durable responses will likely require combination of PARPi with other agents. Citation Format: Olga Kondrashova, Vanessa F. Bonazzi, Deborah Smith, Katia Nones, Asmerom Sengal, Robert Ju, Leisl M. Packer, Lambros T Koufariotis, Stephen H. Kazakoff, Aimee L. Davidson, Priya Ramarao-Milne, Vanessa Lakis, Felicity Newell, Rebecca Rogers, Claire Davies, Naven Chetty, Lewis Perrin, John Pearson, Ann-Marie Patch, Andrea Garrett, Nicola Waddell, Pamela Pollock, James Nicklin. Genomic characterization of endometrial cancer patient-derived xenografts reveals intratumor heterogeneity [abstract]. In: Proceedings of the AACR Virtual Special Conference: Endometrial Cancer: New Biology Driving Research and Treatment; 2020 Nov 9-10. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(3_Suppl):Abstract nr PR007.

Background Declining costs of next-generation sequencing technologies has led to their more widespread use for the identification of targetable mutations. Nonetheless, while multigene panels are being assimilated into clinical practice, the use of whole-exome and whole-genome sequencing (WES and WGS) remains limited, as its added value is unclear in the clinic. Methods In our study, we have used the Cancer Genome Interpreter (CGI) to identify the actionable variants that are detected by WGS and compared this to in-silico down-sampled regions of WES and a hotspot panel. Results We show that within some tumour types, WGS is invaluable in identifying actionable variants that involve copy number defects and structural aberrations. Moreover, WGS successfully identifies many resistance biomarkers that would have otherwise been missed by WES and panels, particularly in the breast cancer dataset studied. WGS is more valuable of the detection of actionable variants in some tumour types, whereas hotspot panels perform well for other tumour types. Additionally, we show that a tumour type-agnostic approach to selecting genomic biomarker-based drug allocation increases the number of possible FDA-approved and clinical trial-based drug prescriptions for any given genomic biomarker in the patient cohorts studied. Conclusions Taken together, we show that WGS has the potential to impact patient care by identifying more targetable mutations and therefore expand drug options available for the patient. Furthermore, we illustrate the potential of tumour type-agnostic drug repurposing in nine cancer datasets. Legal entity responsible for the study QIMR Berghofer Medical Research Institute. Funding QIMR Berghofer Medical Research Institute. Disclosure All authors have declared no conflicts of interest.

We examined transcriptional changes in CD4+ T cells during blood-stage Plasmodium falciparum infection in individuals without a history of previous parasite exposure. Transcription of CXCL8 (encoding interleukin 8) in CD4+ T cells was identified as an early biomarker of submicroscopic P. falciparum infection, with predictive power for parasite growth. Following antiparasitic drug treatment, a CD4+ T-cell regulatory phenotype developed. PD1 expression on CD49b+CD4+ T (putative type I regulatory T) cells after drug treatment negatively correlated with earlier parasite growth. Blockade of PD1 but no other immune checkpoint molecules tested increased interferon γ and interleukin 10 production in an ex vivo antigen-specific cellular assay at the peak of infection. These results demonstrate the early development of an immunoregulatory CD4+ T-cell phenotype in blood-stage P. falciparum infection and show that a selective immune checkpoint blockade may be used to modulate early developing antiparasitic immunoregulatory pathways as part of malaria vaccine and/or drug treatment protocols.